1. Field of the Invention
The present invention relates to a method for detecting a cell having microsatellite instability based on gene expression analysis, which is useful for cancer diagnosis.
2. Background Art
One of the unexpected findings clarified by the human genome project is a report that the number of genes contained in the human genome is much smaller than that has been predicted, and that it is only approximately 25,000. This gene number does not significantly differ from those of lower organisms, and it is insufficient for explanation of the complicated brain system of human and functions of the immune system thereof. Thus, it is considered that various expression mechanisms derived from a single gene play an important role in the complicated biological functions of higher animals.
The variability of the expression of mRNA and a protein from a single gene is mainly controlled by an alternative splicing or a usage of an alternative promoter at the stage of mRNA transcription or RNA processing (see, Non-Patent Document 1). The term “alternative splicing” is used to mean a mechanism for generating several types of mature mRNAs (splicing variants) from a single mRNA precursor as a result of a phenomenon whereby a splicing reaction that generally occurs between splice sites adjacent to each other occurs between various splice sites, when several introns exist in an mRNA precursor. On the other hand, the term “alternative promoter” is used to refer to the presence of several alternative promoters for controlling the transcription of a single gene. When such an alternative promoter exists, several transcription initiation sites are present in the gene. As a result, several types of transcripts having different 5′-terminal sequences are generated. Recently, as a result of searching for 1,780,295 types of full-length cDNA sequences that are contained in an oligo-cap cDNA clones, it has been unexpectedly reported that approximately 52% of genes assigned to the RefSeq database at NCBI, U.S.A. are under the control of alternative promoters, and that 3.1 alternative promoters exist for a single gene on average (see, Non-Patent Document 2). As a result of such a variety of expressions from a single gene, the diversity of organisms or complicated biological functions have been generated in the evolutionary process. On the other hand, it has also been known that such a variety of expressions may cause generation of abnormal proteins or deletion of such a protein itself in individuals, and as a result, it may induce malignant transformation of cells (see, Non-Patent Document 3).
The genetic understanding of hereditary non-polyposis colorectal cancer (HNPCC) has dramatically progressed as a result of discovery of various types of genes associated with mismatch repair. Such a mismatch repair mechanism is a mechanism for recognizing an abnormal base pairing (mismatch base pairs) generated during DNA replication or genetic recombination, and eliminating and repairing the mismatched base pairs. With regard to HNPCC, one of the most typical diseases among familial neoplastic diseases, it has been known that the microsatellite instability (MSI) in the DNA of tumor tissues, which reflects deficiency in a mismatch repair system, is positive in 90% or more of the cases thereof (see, Non-Patent Document 4). The term “microsatellite” is used to mean a repetitive sequence formed by repeating several to several tens of repeat units each consisting of 2 to 5 bases. The term “instability” is used herein to mean a phenomenon in which repeat numbers of microsatellites are abnormally increased or decreased. MSI can be typically detected by assaying microsatellite markers (BAT25, BAT26, D2S123, D5S346, D17S250, etc.) using PCR method. Thus, detection of MSI contributes to HNPCC diagnosis (see, Non-Patent Document 5). In addition, in an international consensus conference regarding MSI detection in 1997, tumor in which instability is detected from two or more markers as mentioned above was defined as MSI-H (a state where 30% or more MSI is detected in tumor), and it was determined that such tumor has clinicopathologic characteristics.
Genetic diagnosis of cancer is based on the results of analysis of mutation of a causative gene in many cases. However, differing from familial adenomatous polyposis (FAP) whose single causative gene is APC, five causative genes have been identified (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6) in the case of HNPCC. However, since the ratio of developing the disease (penetrance rate) when such causative genes are mutated is not 100%, the genetic diagnosis of HNPCC based on mutation of the aforementioned causative genes has not yet reached a clinical level at the present time, and it is considered that such genetic diagnosis is still at a study level in the classification of American Society of Clinical Oncology (ASCO). With regard to cancer-associated genes such as hMLH1, a method, which comprises detecting genetic mutation or methylation on their genomic sequences and detecting cancer using such genetic mutation or methylation as an indicator, has been proposed (Patent Documents 1 and 2). However, the disease cover ratio of this method is not very high, and the method has also been problematic in terms of simplicity and promptness. Hence, it has been desired that a simple and highly reliable method for testing cells, which is useful for the diagnosis of cancers such as HNPCC as a typical example, be developed. If a cell test method capable of screening of cancer at a gene expression level were developed, it means that the real-time detection system in that method would be effective. However, such a useful test method has not yet been developed.    [Patent Document 1] JP Patent Publication (Kokai) No. 2005-304497 A    [Patent Document 2] JP Patent Publication (Kohyo) No. 2002-533061 A    [Non-Patent Document 1] Landry J. R. et al., Trends in Genetics, (2003) 19(11) p. 640-648    [Non-Patent Document 2] Kimura K. et al., Genome Research, (2006) 16: p. 55-65    [Non-Patent Document 3] Brinkman B. M., Clin. Biochem., (2004) 37(7): p. 584-594    [Non-Patent Document 4] Liu B. et al., Nature Med., (1996) 2: p. 169-174    [Non-Patent Document 5] Laiho P. et al., Cancer Research, (2002) 62: p. 1166-1170    [Non-Patent Document 6] Deng G. et al., Cancer Research, (1999) 59: p. 2029-2033